1. Field of the Invention
The present invention provides a method for the detection and sorting of microparticles in a mixture of microparticles. The method of the present invention allows for the detection and sorting of many distinct microparticle classes. Detection and sorting is on the basis of microparticle size, the fluorescence spectrum of any attached reporter molecule, the fluorescence intensity of the reporter molecule, and the number of particles in each classification bin. These microparticle classes have particular applications in many genetic or biochemical multiplexing studies and especially as binding agents for the detection of aneuploidy in an organism or embryo of the organism. In humans, the detection and sorting of at least 24 classes of microparticles would be sufficient for a single tube method for the simultaneous detection of aneuploidy in all chromosomes, wherein each distinct microparticle class comprises a polynucleotide sequence complementary to, and specific for, a polynucleotide sequence that is unique to a particular human chromosome. Furthermore, using currently available technology, the present method has application for the simultaneous detection of aneuploidy in all chromosomes for an organism that has 216 or fewer pairs of chromosomes. Kits for the simultaneous detection of aneuploidy in one or more human chromosomes are also provided.
2. Description of the Prior Art
Bibliographic details of the publications referred to in this specification are also collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Under normal circumstances in a diploid organism, one chromosome from each parent is transmitted to the offspring embryo. However, non-disjunction events, on the maternal, paternal or both sides can lead to embryos with aberrant chromosome number, a condition known as aneuploidy.
Euploidy is the condition of having a correct number of structurally normal chromosomes. For example, euploid human females have 46 chromosomes (44 autosomes and two X chromosomes), whereas euploid bulls have 60 chromosomes (58 autosomes plus an X and a Y chromosome).
Aneuploidy is the condition of having less than or more than the natural diploid number of chromosomes, and is the most frequently observed type of cytogenetic abnormality. In other words, it is any deviation from euploidy, although many authors restrict use of this term to conditions in which only a small number of chromosomes are missing or added.
Generally, aneuploidy is recognized as a small deviation from euploidy for the simple reason that major deviations are rarely compatible with survival, and such individuals usually die prenatally.
The two most commonly observed forms of aneuploidy are monosomy and trisomy.
Monosomy is lack of one of a pair of chromosomes. An individual having only one chromosome 6 is said to have monosomy 6. A common monosomy seen in many species is X chromosome monosomy, also known as Turner's syndrome in humans. Monosomy is most commonly lethal during prenatal development.
Trisomy is having three chromosomes of a particular type. A common autosomal trisomy in humans is Down syndrome, or trisomy 21, in which a person has three instead of the normal two chromosome 21's. Trisomy is a specific instance of polysomy, a more general term that indicates having more than two of any given chromosome (in diploid organisms).
Another type of aneuploidy is triploidy. A triploid individual has three of every chromosome, that is, three haploid sets of chromosomes. A triploid human would have 69 chromosomes (3 haploid sets of 23), and a triploid dog would have 117 chromosomes. Production of triploids seems to be relatively common and can occur by, for example, fertilization by two sperm. However, birth of a live triploid is extraordinarily rare and such individuals are quite abnormal. The rare triploid that survives for more than a few hours after birth is almost certainly a mosaic, having a large proportion of diploid cells.
A chromosome deletion occurs when the chromosome breaks and a piece is lost. This of course involves loss of genetic information and results in what could be considered “partial monosomy” for that chromosome.
A related abnormality is a chromosome inversion. In this case, a break or breaks occur and that fragment of chromosome is inverted and rejoined rather than being lost. Inversions are thus rearrangements that do not involve loss of genetic material and, unless the breakpoints disrupt an important gene, individuals carrying inversions have a normal phenotype.
In a monosomic sample, with 2 n−1 chromosomes, one entire chromosome and all its loci are lost. Similarly, in a 2 n+1 trisomic sample, one extra chromosome is present in each cell, meaning one specific chromosome is represented three times due to a non-dysjunction event, usually in the female gametogenesis. A similar, but more pronounced, situation occurs in the case of a triploid sample in which each chromosome is represented three times instead of twice in each cell.
Pregnancies can be established in infertile women using the technique of in-vitro fertilization (IVF). In spite of the high rate of fertilization in-vitro, the rate of pregnancy following these procedures is relatively low, ranging from 15% to 25%. Cytogenetic studies of human oocytes fixed after failing to fertilize in-vitro display a relatively high incidence of chromosomal abnormalities (aneuploidy). Also, studies of many spontaneous abortions and pre-term embryos show that chromosomal abnormalities may be the main cause of fetal loss. The frequency of chromosomal abnormality in embryos generated using IVF is much higher than total abnormalities reported for sperm and oocytes.
In the IVF procedure, aneuploidy is the most frequently observed abnormality in the embryos generated. Many reports strongly indicate that chromosomal aneuploidy is the prime cause of fertilization failure in oocytes and implantation failure of embryos. Aneuploidy mainly arises during meiotic non-dysjunction; but many environmental factors may also disrupt spindle function and eventually lead to the formation of aneuploid embryos.
Using methods currently known in the art to assess the embryo's gross chromosome makeup, one would perform cytogenetic analyses, such as karyotyping. However, this method is not a practical solution for single cells, and therefore cannot be performed as a pre-implantation screen.
Therefore, there is a need to develop rapid, inexpensive, automatable methods for detecting aneuploidy in an embryo that can be applied in the pre-implantation setting for in-vitro fertilization. The present invention provides a method, which has application, inter alia, as a rapid, single-tube method for the simultaneous detection of aneuploidy in one, multiple or all chromosomes of a subject.
In particular, this method may increase the success rates of IVF, as embryos with aberrant chromosome numbers (aneuploid) could be screened out by a pre-implantation scan of the embryogenic genetic component.